Identification of Streptomyces coelicolor M145 genomic region involved in biosynthesis of teichulosonic acid–cell wall glycopolymer

The cell wall of the model actinomycete Streptomyces coelicolor M145 has recently been shown to contain the novel glycopolymer teichulosonic acid. The major building block of this polymer is 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn), suggesting initial clues about the genetic control of biosynthesis of this cell wall component. Here, through genome mining and gene knockouts, we demonstrate that the sco4879–sco4882 genomic region of S. coelicolor M145 is necessary for biosynthesis of teichulosonic acid. Specifically, mutants carrying individual knockouts of sco4879, sco4880 and sco4881 genes do not produce Kdn-containing glycopolymer and instead accumulate the minor cell wall component poly(diglycosyl 1-phosphate). Our studies provide evidence that this region is at least partly responsible for biosynthesis of Kdn, whereas flanking genes might control the other steps of teichulosonic acid formation.     

Analysis of Streptomyces coelicolor membrane proteome using two-dimensional native/native and native/sodium dodecyl sulfate gel electrophoresis

Analysis of the oligomeric state of a protein may provide insights into its physiological functions. Because membrane proteins are considered to be the workhorses of energy generation and polypeptide and nutrient transportation, in this study we characterized the membrane-associated proteome of Streptomyces coelicolor by two-dimensional (2D) blue native/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), high-resolution clear native/native PAGE, and native/SDS–PAGE. A total of 77 proteins were identified, and 20 proteins belonging to 15 complexes were characterized. Moreover, the resolution of high-resolution clear native/SDS–PAGE is much higher than that of blue native/SDS–PAGE. OBP (SCO5477) and BldKB (SCO5113) were identified as the main protein spots from the membrane fractions of S. coelicolor M145, suggesting that these two proteins are involved in extracellular peptide transportation. These two transporters exhibited multiple oligomeric states in the native PAGE system, which may suggest their multiple physiological functions in the development of S. coelicolor.     

Molecular characterization of SCO0765 as a cellotriose releasing endo-<Emphasis Type="Italic">β</Emphasis>-1,4-cellulase from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A(3)

The sco0765 gene was annotated as a glycosyl hydrolase family 5 endoglucanase from the genomic sequence of Streptomyces coelicolor A3(2) and consisted of 2,241 bp encoding a polypeptide of 747 amino acids (molecular weight of 80.5 kDa) with a 29-amino acid signal peptide for secretion. The SCO0765 recombinant protein was heterogeneously over-expressed in Streptomyces lividans TK24 under the control of a strong ermE* promoter. The purified SCO0765 protein showed the expected molecular weight of the mature form (718 aa, 77.6 kDa) on sodium dodecyl sulfate-polyacryl amide gel electrophoresis. SCO0765 showed high activity toward β-glucan and carboxymethyl cellulose (CMC) and negligible activity to Avicel, xylan, and xyloglucan. The SCO0765 cellulase had a maximum activity at pH 6.0 and 40°C toward CMC and at pH 9.0 and 50–60°C toward β-glucan. Thin layer chromatography of the hydrolyzed products of CMC and β-glucan by SCO0765 gave cellotriose as the major product and cellotetraose, cellopentaose, and longer oligosaccharides as the minor products. These results clearly demonstrate that SCO0765 is an endo-β-1,4-cellulase, hydrolyzing the β-1,4 glycosidic bond of cellulose into cellotriose.     

Analysis of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> M145 genes <Emphasis Type="Italic">SCO4164</Emphasis> and <Emphasis Type="Italic">SCO5854</Emphasis> encoding putative rhodaneses

Streptomyces coelicolor genome carries two apparently paralogous genes, SCO4164 and SCO5854, that encode putative thiosulfate sulfurtransferases (rhodaneses). These genes (and their presumed translation products) are highly conserved and widely distributed across actinobacterial genomes. The SCO4164 knockout strain was unable to grow on minimal media with either sulfate or sulfite as the sole sulfur source. The SCO5854 mutant had no growth defects in the presence of various sulfur sources; however, it produced significantly less amounts of actinorhodin. Furthermore, we discuss possible links between basic interconversions of inorganic sulfur species and secondary metabolism in S. coelicolor.     

Polyhydroxyalkanoate accumulation in Streptomyces coelicolor affected by SCO7613 gene region

Polyhydroxyalkanoate (PHA) is stored as an important carbon and energy source in bacterial cells. For biomedical applications, gram-positive bacteria can be better sources of PHAs, since they lack outer membrane lipopolysaccharide. Although gram-positive Streptomyces coelicolor A3(2) has been indicated as a high potential PHA producer, pha C gene that encodes the key enzyme PHA synthase in the metabolic pathway is not determined in its genome. BLAST search results of the GenBank database argued that SCO7613 could specify a putative polyhydroxyalkanoate synthase (PhaC) responsible for PHA biosynthesis. Deduced amino acid sequence of SCO7613 showed the presence of conserved lipase box like sequence, ⁵⁵⁵GASAG⁵⁵⁹, in which serine residue was present as the active nucleophile. Present study describes deletion of putative S. coelicolor pha C gene via PCR dependent method. We showed that SCO7613 is not an essential gene in S. coelicolor and its deletion affected PHA accumulation negatively although it is not ceased. Transcomplementation abolished the mutant phenotype, demonstrating that the decrease in PHA resulted from the deletion of SCO7613.     

Identification and biochemical characterization of a 5-dimethylallyl tryptophan synthase in Streptomyces coelicolor A3(2)

The genome sequencing of Streptomyces coelicolor A3(2) has lead to the identification of numerous cryptic gene clusters involved in the biosynthesis of secondary metabolites; throwing open the challenge of identifying the enzymatic functions that the gene clusters are associated with. In this work, we report the biochemical characterization of one such cryptic gene, SCO7467 from S. coelicolor A3(2), which is annotated as a prenyltransferase. Based on LC–MS and 2D-NMR studies, we show that SCO7467 acts as a 5-dimethylallyl tryptophan synthase (5-DMATS), and catalyzes the transfer of a dimethylallyl group to the C-5 position of the indole ring of l-tryptophan. The studies indicate that SCO7467 could be involved in the synthesis of C-5 prenylated indole alkaloids, which may exhibit unique pharmacological and biological properties.     

A possible extended family of regulators of sigma factor activity in Streptomyces coelicolor

SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific σ^F, suggesting that it is an antagonistic regulator of σ^F. Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-σ^F during a subsequent global yeast two-hybrid screen, and the SCO0869-SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA of B. subtilis. It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.     

Proteome analysis of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> mutants affected in the proteasome system reveals changes in stress-responsive proteins

Prokaryotic 20S proteasomes are confined to archaebacteria and actinomycetes. Bacterial targets of this compartmentalized multi-subunit protease have not yet been identified and its physiological function in prokaryotes remains unknown. In this study, intracellular and extracellular proteomes of Streptomyces coelicolor A3(2) mutants affected in the structural genes of the 20S proteasome, in the gene encoding the presumed proteasome-accessory AAA ATPase ARC, or in two putative proteasome-associated actinomycete-specific genes (sco1646, sco1647) were analysed, revealing modified patterns of stress-responsive proteins. In addition, the extracellular protease profile of the sco1647 mutant was significantly altered. The most prominent change, common to the four mutants, was a strongly increased level of the non-heme chloroperoxidase SCO0465, coinciding with an increased resistance to cumene hydroperoxide. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Novel Tryptophan Metabolism by a Potential Gene Cluster That Is Widely Distributed among Actinomycetes

The characterization of potential gene clusters is a promising strategy for the identification of novel natural products and the expansion of structural diversity. However, there are often difficulties in identifying potential metabolites because their biosynthetic genes are either silenced or expressed only at a low level. Here, we report the identification of a novel metabolite that is synthesized by a potential gene cluster containing an indole prenyltransferase gene (SCO7467) and a flavin-dependent monooxygenase (FMO) gene (SCO7468), which were mined from the genome of Streptomyces coelicolor A3(2). We introduced these two genes into the closely related Streptomyces lividans TK23 and analyzed the culture broths of the transformants. This process allowed us to identify a novel metabolite, 5-dimethylallylindole-3-acetonitrile (5-DMAIAN) that was overproduced in the transformant. Biochemical characterization of the recombinant SCO7467 and SCO7468 demonstrated the novel l-tryptophan metabolism leading to 5-DMAIAN. SCO7467 catalyzes the prenylation of l-tryptophan to form 5-dimethylallyl-l-tryptophan (5-DMAT). This enzyme is the first actinomycetes prenyltransferase known to catalyze the addition of a dimethylallyl group to the C-5 of tryptophan. SCO7468 then catalyzes the conversion of 5-DMAT into 5-dimethylallylindole-3-acetaldoxime (5-DMAIAOx). An aldoxime-forming reaction catalyzed by the FMO enzyme was also identified for the first time in this study. Finally, dehydration of 5-DMAIAOx presumably occurs to yield 5-DMAIAN. This study provides insight into the biosynthesis of prenylated indoles that have been purified from actinomycetes.